User Guide

Watchmaker mRNA Library Prep Kit

Table of Contents

Product Description

Product Description. . . . . . . . . . . . . . . . . . . . . 1

The Watchmaker mRNA Library Prep Kit is designed for the highly
streamlined preparation of stranded mRNA sequencing libraries
from 2.5 ng to 1000 ng intact total eukaryotic RNA with high librarycomplexity and low coverage bias.

Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

The Watchmaker mRNA Library Prep Kit enriches for mature,polyadenylated mRNAs and converts those mRNAs to adapterligated, double-stranded cDNA fragments through a small numberof chemical and enzymatic manipulations:

Product Applications. . . . . . . . . . . . . . . . . . . . 2
Storage and Handling . . . . . . . . . . . . . . . . . . . 2
Workflow Overview . . . . . . . . . . . . . . . . . . . . . 2
Required Materials not Included . . . . . . . . . . 3
Prior to Starting . . . . . . . . . . . . . . . . . . . . . . . . 3• Highly speciﬁc capture of full-length polyadenylated (poly(A))
RNA transcripts such as eukaryotic mRNA and some noncoding RNAs

Library Construction Protocol . . . . . . . . . . . . 5• RNA fragmentation and priming for 1st strand cDNA synthesis

Revision History . . . . . . . . . . . . . . . . . . . . . . . 12

Appendix A: Amplification withParamagnetic Purification Beads . . . . . . . . 12

• 1st strand cDNA synthesis leveraging a speciﬁcally engineered
reverse transcriptase• Combined 2nd strand cDNA synthesis and A-tailing, during
which dUTP is incorporated to maintain strand information• Adapter ligation, where truncated, full-length, or custom
adapters utilizing a 3' T overhang are ligated to fragments• Library ampliﬁcation with Equinox™ Ampliﬁcation Master Mix(2X) for high-ﬁdelity and high-eﬃciency PCR

Kit Contents
Bundled kit

Kit

WatchmakermRNA Capture Kit

Kit code

7K0105-024 (24 rxn)
7K0105-096 (96 rxn)

WatchmakermRNA Library Prep Kit
7BK0001-024 (24 rxn)
7BK0001-096 (96 rxn)

Watchmaker
RNA Library Prep Kit

7K0078-024 (24 rxn)
7K0078-096 (96 rxn)

Description

Component Volume
24 rxn kit

96 rxn kit

mRNA Capture Beads

270 µL

1.2 mL

Bead Prep Buffer

2.8 mL

12 mL

Capture Buffer

9.5 mL

37 mL

Final Wash Buffer

6.8 mL

27 mL

Frag & Prime Buffer

290 µL

1.3 mL

1st Strand Buffer

240 µL1.08 mL

1st Strand Enzyme

30 µL

120 µL

2nd Strand Buffer

370 µL

1.68 mL

2nd Strand Enzyme

30 µL

120 µL

Ligation Buffer

1.06 mL

4.8 mL

Ligation Enzyme

140 µL

600 µL

Equinox Ampliﬁcation Master Mix (2X)

690 µL

3 mL

P5/P7 Primer Mix (10X)

144 µL

600 µLFFPE Treatment Buffer1

140 µL

600 µLThe FFPE Treatment Buffer is not required or recommended for mRNA library preparation. When used on its own or in conjunction with the Polaris™ Depletion Kit, the Watchmaker
RNA Library Prep Kit is compatible with the FFPE samples.

1

For custom formats, contact the Sales Team at sales@watchmakergenomics.com.
For Research Use Only. Not for use in diagnostic procedures.

v1.1.0823

Watchmaker mRNA Library Prep Kit

Product Applications
This workﬂow was developed to address the highly speciﬁc
needs of mRNA sequencing and the associated areasof variant calling, isoform and gene fusion identiﬁcation,and gene expression analysis. These applications require
high library complexity, low bias, uniform coverage, andminimal experimental variability in order to support robust
sensitivity, speciﬁcity, and reproducibility.
This library preparation kit is ideally suited for:

User Guide

Workflow Overview
mRNA Capture Bead
Preparation
10-minute hands-on time

RNA Sample (50 µL)
Suspended in RNase-free water• Gene expression analysis• Isoform/splice variant/gene fusion identiﬁcation• Single nucleotide variant detection• Novel transcript discovery• Workﬂows that employ unique molecular identiﬁers(UMIs) for improved sensitivity

1st and 2nd poly(A)RNA Capture
25- to 45-minute hands-on time
2X 7-minute incubation

Storage and Handling

Fragmentation
and Priming (25 µL)
5- to 15-minute hands-on time
Up to 10-minute incubation

The Watchmaker mRNA Library Prep Kits are shipped oncold packs. Upon receipt, store the Watchmaker mRNA
Capture Kit (K0105) at 4°C and the Watchmaker RNA
Library Prep Kit (K0078) at -20 ± 5°C.

1st Strand Synthesis (35 µL)
5- to 15-minute hands-on time
40-minute incubation

Some components are viscous; therefore, take care tohomogenize solutions thoroughly before use and during
reaction setup. SPRI beads should be handled as per the
manufacturer’s guidelines.
Buffers in the Watchmaker mRNA Capture Kit should
be mixed by inverting the tubes and NOT by vortexing
to prevent foaming. All buffers in the Watchmaker RNA
Library Prep Kit should be vortexed for at least 5 sec
before use.
Enzymes and the Equinox Ampliﬁcation Master Mix (2X)
should be inverted ten times prior to use. The 1st Strand
Buffer is photosensitive. Keep it in the kit box until thawing
is required. Take care to protect it from direct sunlightwhile thawing and in use.
All master mixes (buffer and enzyme combined) preparedin the protocol should be stored at 4°C unless statedotherwise. The master mixes should be stable for up to
24 hours at 4°C.

2nd Strand Synthesis
and A-Tailing (50 µL)
5- to 15-minute hands-on time
15-minute incubation

Adapter Ligation (100 µL)
5- to 15-minute hands-on time
15-minute incubation

Post-ligation Cleanup (170 µL)
20-minute hands-on time
SPRI 0.7X

Library Ampliﬁcation (50 µL)
5- to 15-minute hands-on time
Variable incubation time

Post-ampliﬁcation
Cleanup (100 µL)
20-minute hands-on time
SPRI 1.0X or 0.7X

For Research Use Only. Not for use in diagnostic procedures.

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Watchmaker mRNA Library Prep Kit

User Guide

Required Materials not Included

Prior to Starting• Adapters (see Prior to Starting)

Input RNA Quality and Quantity• Adapter diluent (e.g., 10 mM Tris-HCl, pH 8.0, 10 mM
NaCl)

This kit is compatible with intact (RIN >7) total RNA
samples suspended in 50 µL of RNase-free water.

• Magnetic rack compatible with 0.2 mL PCR tubes and/or 96-well plate

High-quality total RNA ranging from 2.5 – 1000 ng has
been tested and shown to produce high-performing
libraries.

• Magnetic rack compatible with 1.5 – 2 mL microtubes• Ampure® XP Beads (Beckman Coulter, Inc. #A63881)
or equivalent• 80% Ethanol• 200 µL thin-wall PCR tubes compatible withthermocycler, or 96-well 0.2 mL PCR Plates and PlateSeal

RNA should be accurately quantiﬁed by Qubit® Fluorometeror similar prior to starting.
Assess total RNA quality via an electrophoretic method,such as Agilent BioAnalyzer or TapeStation.

• 10 mM Tris-HCl, pH 8.0

ERCC RNA Spike-In Controls are not recommended for use
with the Watchmaker mRNA Library Prep Kit due to theirshort poly(A) tails (20 – 26 nt long), which do not imitatetypical polyadenylated RNA transcripts. If ERCC RNA
Spike-In Controls are included, a lower number of reads
mapping to ERCCs is expected due to lower efficiency
hybridization to the oligo dT.

• Thermocycler

Input RNA Purity• Vortex mixer and/or plate vortexer

RNA inputs should be free from contaminating DNA.
If the total RNA contains DNA, remove the contamination
by incubating with DNase I (not supplied with kit). Residual
DNase I may interfere with library preparation, so it isimportant to ensure no residual enzyme remains in thesample.

• 1 mL, 2 mL, 5 mL microtubes (RNase-free)• RNase-free water• Plate centrifuge and/or benchtop picofuge• Fragment Analyzer™, Bioanalyzer®, TapeStation(Agilent Technologies, Inc.), or similar instrument andconsumables
These protocols are designed for use with the specified
labware, consumables, and calibrated equipment.

RNA should be suspended in RNase-free water and be freeof salts (e.g., Mg2+, or guanidinium salts), chelating agents(e.g., EDTA or EGTA), and organics (e.g., phenol or ethanol).
RNA Handling
To avoid RNase contamination, work in a laminar ﬂow
hood, if available, and keep all sample and reagent tubes
closed unless in use. Wear gloves when handling reagentsand preparing libraries. Change gloves and pipette tips if
they come into contact with non-sterile surfaces.
To avoid RNA degradation, store RNA in an RNase-freediluent and limit the number of sample freeze-thaw cycles.
Insert Size
The standard protocol targets ~200 bp inserts.1
When targeting ~300 bp inserts, add 2 extra cycles of
PCR ampliﬁcation (Step 10) and make use of a 0.7X postampliﬁcation SPRI cleanup (Step 11.2).
Mixing Considerations
The Watchmaker mRNA Library Prep Kit has beendeveloped to provide robust and reproducible data whenemploying either vortex or pipette mixing.

As assessed by Illumina NovaSeq (Picard mean insert size)

1

For Research Use Only. Not for use in diagnostic procedures.

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Watchmaker mRNA Library Prep Kit

User Guide

It is recommended to make use of vortexing (2,000 rpm on
an appropriate vortex mixer) where possible as it provides
uniform and reproducible mixing of samples. Tubes
and/or plates should be sealed prior to vortexing andbrieﬂy centrifuged after vortexing. Extreme care should
be taken to ensure that no residual droplets remain on theside or at the top of tubes and/or plates after centrifuging.

sequencing workﬂows, a uniquely barcoded primer mix
will be required (and must be added individually) for eachlibrary to be sequenced on the same ﬂow cell.

For automation, or if access to an appropriate vortex mixer
is not available, use pipette mixing. Samples should be
pipetted carefully to minimize foaming and tubes/plates
brieﬂy centrifuged after mixing.

Primers may also incorporate chemical modiﬁcations(e.g., one or more 3'-phosphorothioate bonds) to improvespeciﬁcity.

Adapters
This kit is compatible with adapters that have a
3' overhanging T to facilitate adapter orientation during
dscDNA ligation. Note that adapter quality impacts overall
library preparation eﬃciency. Ensure that adapters areadequately duplexed and at the appropriate concentration
prior to use.
When using ‘stubby’, adapters where sample indexes
are added during subsequent library ampliﬁcation, usersupplied, uniquely indexed PCR primers will be required
for the ampliﬁcation of each library to be sequenced on
the same ﬂow cell.
Stubby adapters provide improved library prep
eﬃciency due to the ability to include them at increased
concentrations in the ligation reaction. We strongly
recommend the use of stubby adapters for maximum
performance.
This workﬂow is also compatible with full-length adapterswhere sample indexes are added during ligation. Whenusing full-length adapters, a unique sample index isrequired for all samples to be sequenced on the same ﬂow
cell. Refer to the technical documentation provided by theadapter vendor for recommendations on optimal pooling.

Primers should always use equimolar concentrations of theforward and reverse primers. A primer pre-mix containing
20 µM of each primer (resulting in a ﬁnal concentration of
2 µM each in the ampliﬁcation reaction) is recommended.

Use a buffered solution, such as 10 mM Tris-HCl, pH 8.0,
to store and dilute primers. Limit the number of freezethaw cycles.
Library Ampliﬁcation Optimization
Annealing Temperature
For the truncated adapter scheme detailed in Glenn, et.
al. 2019,2 use an annealing temperature of 55°C. For otherprimers, an annealing temperature gradient (55°C to 70°C)
may be performed to determine the optimal condition for
ampliﬁcation.
Extension TimeLonger extension times may be employed to ensure
eﬃcient ampliﬁcation of longer-insert libraries. A 30sec extension is suﬃcient for libraries with a modefragment size up to 500 bp; a 45-sec extension time isrecommended for libraries with mode fragment sizes>500 bp. The optimal condition for each application may
have to be determined empirically.
Cycle Number

Contact support@watchmakergenomics.com for moredetails and our Adapter Recommendations Guide.

This protocol provides a starting point for PCR cycle
number optimization based on RNA input into library
preparation and insert size. Adapter-ligated libraries may
be quantiﬁed by qPCR or estimated by other means or
methods to determine the optimal number of ampliﬁcation
cycles for the desired library yield.

P5/P7 Ampliﬁcation Primers

SPRI Puriﬁcation Beads

The P5/P7 Primer Mix (10X) is supplied at a concentrationof 20 µM of each primer and is appropriate for theampliﬁcation of full-length adapter-ligated libraries.
P5: AATGATACGGCGACCACCGA
P7: CAAGCAGAAGACGGCATACGAGAT
User-supplied Ampliﬁcation Primers
When using truncated, or ‘stubby’, adapters in multiplexed2

The protocol outlined below assumes the use of AMPure
XP (Beckman Coulter) reagent for bead puriﬁcation steps.
Other SPRI bead brands may be used, assuming they are
nuclease-free and deliver equivalent sizing performance at
the given bead ratios. Otherwise, bead puriﬁcation ratios
will need to be optimized for the bead brand used.
Ensure beads are equilibrated to room temperature andthoroughly resuspended via vortexing prior to use.

Glenn TC, Nilsen RA, Kieran TJ, et al. Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatoriallyindexed Illumina libraries (iTru & iNext). PeerJ. 2019;7:e7755. Published 2019 Oct 11. doi:10.7717/peerj.7755]

For Research Use Only. Not for use in diagnostic procedures.

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Watchmaker mRNA Library Prep Kit

Library Construction Protocol
Recommendations• All mRNA Capture components should be equilibrated
to room temperature for 20 minutes and inverted
several times before use.
NOTE: Do NOT vortex Watchmaker mRNA Capture Kit
buffers.
• Ensure all Watchmaker RNA Library Prep Kit buffers
are fully thawed on ice before use. Once thawed, invert
the tubes several times, and vortex for at least 5 sec toensure the reagent is fully mixed.
• The Fragmentation & Priming Master mix (Step 4.1)
may be prepared ahead of time and stored on ice
before use.
• Place enzymes and the Equinox Ampliﬁcation MasterMix (2X) on ice before use. Invert the tubes 10 times
to mix.
• Vortexing is recommended for reaction mixing
throughout. Where possible, centrifuge brieﬂy to
remove any excess liquid from the tubes and collect all
liquid in the tube lids prior to opening them—see Prior
to Starting: Mixing Considerations.
• We recommend making master mixes for eachenzymatic reaction step with a 10% excess to accountfor loss during pipetting.
• Ensure SPRI puriﬁcation beads are fully equilibrated
to room temperature and thoroughly resuspended by
vortexing prior to use.
1. RNA and mRNA Capture Bead Preparation
1.1 After thawing on ice, dilute the total RNA sample
to a ﬁnal volume of 50 µL with RNase-free water.
1.2 Vortex mRNA Capture Beads until all beads arewell resuspended (5 – 10 secs).
1.3 Prepare the mRNA Capture Beads in bulk by
aliquoting 10.5 µL of beads (10 µL + 5% excess)per reaction into a microtube.
NOTE: A recommended maximum of 28 or
36 reactions can be prepared in a 1.5 mL and2 mL tube, respectively. Multiple tubes, or larger
microtubes, may be required depending on thenumber of reactions.

For Research Use Only. Not for use in diagnostic procedures.

User Guide

1.4 Place the tube on the magnetic rack at room
temperature for 2 min or until the solution is clear.
1.5 Remove and discard all of the storage buffer fromthe tube. Take care not to disturb the beads.
1.6 Remove the tube from the magnetic rack and keep
at room temperature.
1.7 Wash the beads by adding 52.5 µL of Bead
Prep Buffer per reaction to the bulk beads from
Step 1.6. Pipette mix at the highest feasible
volume ≥10 times to fully resuspend the beads.
Take care to avoid foaming.
1.8 Place the tube on the magnetic rack at room
temperature for 2 min or until the solution is clear.
1.9 Remove and discard all of the Bead Prep Bufferfrom the tube. Take care not to disturb the beads.
1.10 Remove the tube from the magnetic rack and keep
at room temperature.
1.11 Add 52.5 µL of Bead Prep Buffer per reaction to thebulk beads from Step 1.10. Pipette at the highest
feasible volume ≥10 times to fully resuspend thebeads. Take care to avoid foaming.
1.12 Store the washed mRNA Capture Beads at room
temperature and proceed to 1st Poly(A) RNA
Capture.
NOTE: Washed beads from Step 1.11 can be stored
in the Bead Prep Buffer for up to 1 week at 4°C.
Ensure beads are fully resuspended after storage
before continuing.
2. 1st Poly(A) RNA Capture
2.1 Program a thermocycler as indicated below andinitiate the run to heat the block:
NOTE: This thermocycler program is used again
in Step 3.1.
Step
Lid temperature

Temperature

Time

80°C

N/A

HOLD

25°C

HOLD

Denaturation

75°C

2 min

Hybridization

25°C

5 min

HOLD

25°C

HOLD

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Watchmaker mRNA Library Prep Kit

User Guide

3. 2nd Poly(A) RNA Capture

2.2 To each tube, add the following:
Component

Volume (µL)

RNA sample

50

Washed mRNA Capture Beads(from Step 1.12)50

2.3 Mix on an appropriate vortex mixer (2,000 rpm) for
5 sec. Brieﬂy centrifuge to collect all liquid in thebottom of the tubes.
WORKFLOW: If pipette mixing, mix the reaction
10 times with a pipette set to 80 µL. Brieﬂycentrifuge if possible. See Prior to Starting: Mixing
Considerations.
2.4 Place the tubes into the preheated thermocycler(programmed and initiated in Step 2.1). Advancethe thermocycler from the initial 25°C hold.
2.5 Upon completion of thermocycling, place thetubes on the magnetic rack at room temperaturefor 2 min, or until the beads have collected on theside of the tubes.
2.6 While on the magnet, remove and discard all of thesupernatant from each tube. Avoid disturbing thebeads.
2.7 While on the magnet, and without disturbing thebeads, gently add 180 µL Capture Buffer to thetubes. If using a 0.3 mL tube, then use 250 µL to
fill tube capacity. This is to remove any remaining
uncaptured RNA adsorbed to the tubes.
2.8 While still on the magnet, remove and discard all of
the supernatant from each tube. Avoid disturbing
the beads.
2.9 Remove the tubes from the magnetic rack and add
100 µL of Capture Buffer to each tube.
2.10 Mix on an appropriate vortex mixer (2,000 rpm) for
5 sec to resuspend the beads. Brieﬂy centrifuge tocollect all liquid in the bottom of the tubes.
WORKFLOW: If pipette mixing, mix the reaction 10
times with a pipette set to 80 µL to fully resuspend
the beads in the Capture Buffer and to avoid
excessive foaming. Brieﬂy centrifuge if possible.
See Prior to Starting: Mixing Considerations.
2.11 Proceed immediately to 2nd Poly(A) RNA Capture.

For Research Use Only. Not for use in diagnostic procedures.

3.1 Program a thermocycler as indicated below andinitiate the run to heat the block:
Step

TemperatureTime

Lid temperature

80°C

N/A

HOLD

25°C

HOLD

Denaturation

75°C

2 min

Hybridization

25°C

5 min

HOLD

25°C

HOLD

3.2 Place the tubes into the preheated thermocycler(programmed and initiated in Step 3.1). Advancethe thermocycler from the initial 25°C hold.
3.3 Upon completion of thermocycling, place thetubes on the magnetic rack at room temperaturefor 2 min.
3.4 Remove and discard all of the supernatant fromeach tube. Avoid disturbing the beads.
3.5 While on the magnet, and without disturbing thebeads, gently add 180 µL Final Wash Buffer to thetubes. If using a 0.3 mL tube, then use 250 µL to
ﬁll tube capacity. This is to remove any remaining
uncaptured RNA adsorbed to the tubes.
3.6 While on the magnet, remove and discard all of thesupernatant from the tubes. Avoid disturbing thebeads.
NOTE: Any remaining Final Wash Buffer in the
tubes will dilute the poly(A) RNA proceeding into
Library Preparation, but will not negatively impact
the Library Preparation chemistry.
3.7 Remove the tubes from the magnetic rack andproceed immediately with Fragmentation andPriming.
4. Fragmentation and Priming
4.1 Prepare the Frag & Prime Master Mix as follows foreach reaction:
Component

Volume (µL)

Frag & Prime Buffer

10.8

RNase-free water

16.2

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Watchmaker mRNA Library Prep Kit

4.2 Mix on a touch vortex mixer (2,000 to 3,000 rpm)
for 4 sec. Brieﬂy centrifuge to collect all liquid in
the bottom of the tube.
4.3 Add 27 µL of the Frag & Prime Master Mix to thebeads from Step 3.7.
4.4 Mix on an appropriate vortex mixer (2,000 to
3,000 rpm) for 4 sec to resuspend the beads.
Brieﬂy centrifuge to collect all liquid in the bottomof the tubes.
WORKFLOW: If pipette mixing, mix the reaction
10 times with a pipette set to 25 µL to resuspend
the beads in the Frag & Prime Master Mix. Brieﬂycentrifuge if possible. See Prior to Starting: Mixing
Considerations.
4.5 Program a thermocycler as indicated below, andinitiate the run to hold at 4°C:

User Guide

5. 1st Strand Synthesis
NOTE: The 1st Strand Buffer is photosensitive.
Keep it in the kit box until thawing is required. Takecare to protect it from direct sunlight while thawing
and in use.
5.1 Program a thermocycler as indicated below andinitiate the run to cool the block:
Step

TemperatureTime

Lid temperature

105°C

N/A

Pre-cooling

4°C

HOLD

25°C

10 mincDNA synthesis

42°C

15 minRT inactivation

70°C

15 min

HOLD

4°C

HOLD

NOTE: Insert size is adjusted through modulating
the Post-ampliﬁcation SPRI ratio (Step 11.2)
and ampliﬁcation cycles (Step 10). Irrespectiveof insert size required, the RNA Fragmentation
conditions remain the same. See Prior to Starting:Insert Size for more details.

5.2 For each reaction, prepare the 1st Strand MasterMix as follows on ice:Step

5.3 Mix on a touch vortexer (2,000 to 3,000 rpm) for
4 sec. Brieﬂy centrifuge to collect all liquid in thebottom of the tubes.

TemperatureTime

Lid temperature

105°C

N/A

Pre-cooling

4°C

HOLD

RNA Fragmentation

85°C

10 minHOLD (Priming)

12°C

HOLD

4.6 Place the tubes into the preheated thermocycler.
Advance the thermocycler from the initial 4°C holdto start the RNA Fragmentation incubation.
4.7 After the program has ﬁnished and the samples
have returned to 12°C, place the tubes on amagnet for at least 2 min, or until all beads havebeen collected on the tube walls and the solution
is clear.
4.8 Carefully remove 25 μL of the supernatant, transfer
to fresh, labeled tubes, and proceed immediately
to 1st Strand Synthesis.
NOTE: Take care not to disturb the beads, as beadcarryover can interfere with 1st Strand Synthesis.
Safe stopping point. Samples can be frozen at-20°C for <24 hours.

For Research Use Only. Not for use in diagnostic procedures.

Component

Volume (µL)

1st Strand Buffer

9

1st Strand Enzyme

1

5.4 To each tube, add the 1st Strand Master Mix as
speciﬁed below on ice:
Component

Volume (µL)

Fragmented RNA (from Step 4.8)25

1st Strand Master Mix

10

5.5 Mix on an appropriate vortex mixer (2,000 to
3,000 rpm) for 4 sec. Brieﬂy centrifuge to collect
all liquid in the bottom of the tubes.
5.6 Place reactions in the chilled thermocycler(programmed and initiated in Step 5.1). Advancethe thermocycler from the initial 4°C hold to startthe 25°C incubation.
5.7 Once the 1st Strand Synthesis reaction has
completed, place the tubes on ice or leave in thethermocycler at 4°C. Proceed immediately to 2nd
Strand Synthesis and A-Tailing.

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Watchmaker mRNA Library Prep Kit

User Guide

7. Adapter Ligation

6. 2nd Strand Synthesis and A-Tailing
6.1 Program a thermocycler as indicated below andinitiate the run to cool the block:
Step

Temperature

Time

NOTE: See Prior to Starting for considerations in
adapter selection and design.
7.1 Program a thermocycler as indicated below:

Lid temperature

80°C

N/A

4°C

Step

TemperatureTime

Pre-cooling

HOLD

OFF

N/A

2nd Strand Synthesis

42°C

Lid temperature

5 min

62°C

Pre-cooling

4°C

HOLD

A-Tailing

10 min4°C

Ligation

20°C

15 min

HOLD

HOLD

HOLD

4°C

HOLD1

6.2 For each reaction, prepare the 2nd Strand MasterMix as follows on ice:
Component

Maintain samples on ice following ligation to reduce adapter-dimer
formation prior to Post-ligation Cleanup.

1

7.2

Vortex the thawed Ligation Buffer for 20 sec tofully homogenize the solution before centrifuging
to collect all of the solution at the bottom of thetubes and placing it on ice.

7.3

Using an appropriate diluent (e.g., 10 mM Tris- HCl,
pH 8.0, 10 mM NaCl), prepare the required volumeof each adapter at the concentration speciﬁed ineither Table 1 or Table 2 based on adapter design.
5 µL of adapter at the appropriate concentration isrequired per ligation reaction.

Volume (µL)

2nd Strand Buffer14

2nd Strand Enzyme

1

6.3 Mix on a touch vortexer (2,000 to 3,000 rpm) for
4 sec. Brieﬂy centrifuge to collect all liquid in thebottom of the tubes.
6.4 To each tube, add the 2nd Strand Master Mix as
speciﬁed below on ice:
Component

NOTE: Storing adapter solutions at concentrations<10 µM for extended periods of time is notrecommended.

Volume (µL)

1st Strand Synthesis product35

2nd Strand Master Mix

15

Table 1. Full-length adapter concentration by RNA input
into library prep

6.5 Mix on an appropriate vortex mixer (2,000 to
3,000 rpm) for 4 sec. Brieﬂy centrifuge to collect
all liquid in the bottom of the tubes.

RNA input (ng)≤50Adapter concentration (µM)

1

51 – 150

6.6 Place reactions in the chilled thermocycler(programmed and initiated in Step 6.1). Advancethe thermocycler from the initial 4°C hold to startthe 42°C incubation.

151 – 1000

0.3
1

A second post-ligation cleanup (Step 9) is recommended for inputs of
50 ng and lower when using full-length adapters.

1

6.7 Proceed to adapter ligation after the program has
ﬁnished and the samples have returned to 4°C.
Proceed immediately to Adapter Ligation.

Table 2. Truncated (‘stubby’) adapter concentration by
RNA input into library prep
RNA input (ng)≤10Adapter concentration (µM)

1

11 – 250
251 – 1000

1
4

A second post-ligation cleanup (Step 9) is recommended for inputs of
10 ng and lower when using ‘stubby’ adapters.

1

7.4

For Research Use Only. Not for use in diagnostic procedures.

Add 5 µL of appropriately diluted adapter to eachtube (Step 6.7).

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Watchmaker mRNA Library Prep Kit

7.5

Prepare the Ligation Master Mix as follows:

User Guide

Component

Volume (µL)

Ligation Buffer40

8.8 Incubate tubes at room temperature for at least
30 sec without removing them from the magnet
or disturbing the bead pellet. Carefully remove anddiscard the ethanol.

Ligation Enzyme

5

8.9 Repeat Steps 8.7 – 8.8, for a total of two washes.

7.6

Mix on a touch vortexer (2,000 or 3,000 rpm) for
4 sec. Brieﬂy centrifuge to collect all liquid in thebottom of the tubes.

OPTIONAL: Tubes can be brieﬂy centrifuged topull down excess ethanol prior to removal with a
P10 pipette.

7.7

To each tube, add the Ligation Master Mix as
speciﬁed below on ice:

8.10 Allow remaining ethanol to evaporate by allowing
the pellets to air dry for 3 – 5 min.

Component

7.8

7.9
7.10

Volume (µL)

2nd Strand Synthesis productand adapter

55

Ligation Master Mix

45

The Ligation Master Mix is viscous. Carefully
pipette a minimum volume of 80 µL up and down aminimum of 10 times. Brieﬂy centrifuge if needed
to collect all liquid in the bottom of the tubes.
Place the tubes in the thermocycler and initiate
the program (programmed in Step 7.1).
Once the program has completed, proceed
immediately to Post-ligation Cleanup.

8. Post-ligation Cleanup
8.1 Freshly prepare at least 0.4 mL of an 80% ethanol
solution for each reaction.
8.2 Vortex room temperature SPRI beads to thoroughly
mix. Add 70 µL (0.7X) of room temperature
resuspended beads to each ligation reaction.
8.3 Mix on an appropriate vortex mixer (2,000 or
3,000 rpm) for 4 sec. Brieﬂy centrifuge to collect
all liquid in the bottom of the tubes.
8.4 Incubate the library-bead mixtures at room
temperature for at least 5 min to maximize libraryrecovery.
8.5 Place tubes on a magnet for at least 5 min, or until
all beads have been collected on the tube walls
and the solution is clear.
8.6 Carefully remove and discard the supernatantfrom each tube.
8.7 Add 200 µL of freshly prepared 80% ethanol toeach tube, taking care to not disturb the beadpellet on the tube walls.

For Research Use Only. Not for use in diagnostic procedures.

NOTE: Beads have been suﬃciently dried whencracks appear in the surface of the bead pellet
and no liquid is visible in the tubes. Residual trace
ethanol in the library ampliﬁcation reaction may
decrease performance.
8.11 Remove tubes from the magnet and carefully
resuspend each bead pellet in 22 µL of 10 mM
Tris-HCl, pH 8.0.
8.12 Incubate tubes at room temperature for at least
2 mins before placing back on the magnet.
8.13 Leave tubes on the magnet for at least 2 min, or
until all beads have collected on the tube walls andthe solution is clear.
8.14 Carefully transfer 20 µL of each library-containing
supernatant to a new, labeled tube.
Safe stopping point. Samples can be stored at 4°C
for <24 hours or frozen at -20°C for up to 4 weeks.
9. 2nd Post-ligation Cleanup (Optional)
NOTE: This is an optional step and only
recommended when working with:•

Full-length adapters and equal to or lower than
50 ng of RNA.

•

Truncated (‘stubby’) adapters and equal to or
lower than 10 ng of RNA.

9.1 Freshly prepare at least 0.4 mL of an 80% ethanol
solution for each reaction.
9.2 Vortex room temperature SPRI beads to thoroughly
mix. Add 20 µL (1X) of room temperature
resuspended beads to each eluted library from
Step 8.14.
9.3 Mix on an appropriate vortex mixer (2,000 to
3,000 rpm) for 4 sec. Brieﬂy centrifuge to collect
all liquid in the bottom of the tubes.

9Watchmaker mRNA Library Prep Kit

9.4 Incubate the library-bead mixtures at room
temperature for at least 5 min to maximize libraryrecovery.

User Guide

10. Library Ampliﬁcation and Strand Selection

9.5 Place tubes on a magnet for at least 5 min, or until
all beads have been collected on the tube walls
and the solution is clear.
9.6 Carefully remove and discard the supernatantfrom each tube.
9.7 Add 200 µL of freshly prepared 80% ethanol toeach tube, taking care to not disturb the beadpellet on the tube walls.
9.8 Incubate tubes at room temperature for at least 30
sec without removing the tubes from the magnet
or disturbing the bead pellet. Carefully remove anddiscard the ethanol.

•

If your workﬂow requires library ampliﬁcationin the presence of paramagnetic beads, refer
to Appendix A for bead compatibility.

10.2 Program a thermocycler as indicated below:
Step1

OPTIONAL: Tubes can be brieﬂy centrifuged topull down excess ethanol prior to removing with ap10 pipette.

Temperature

Time

Cycles

Initialdenaturation

98°C

45 sec1

Denaturation

98°C

15 secAnnealing

9.10 Allow remaining ethanol to evaporate by allowing
the pellets to air dry for 3 – 5 min.

9.11 Remove tubes from the magnet and carefully
resuspend each bead pellet in 22 µL of 10 mM
Tris-HCl, pH 8.0.

Library ampliﬁcation is required for strandspeciﬁc sequencing regardless of adapter
conﬁguration used.

10.1 Thaw and equilibrate the Equinox Ampliﬁcation
Master Mix (2X) on ice. Once thawed, invert several
times or swirl vigorously to mix (DO NOT VORTEX).

9.9 Repeat Steps 9.7 – 9.8 for a total of two washes.

NOTE: Beads have been suﬃciently dried whencracks appear in the surface of the bead pellet
and no liquid is visible in the tubes. Residual trace
ethanol in the library ampliﬁcation reaction may
decrease performance.

•

P5/P7
primers: 60°C2
Indexedprimers: 55°C3

30 secExtension

72°C

30 – 45 sec

Finalextension

72°C

60 sec

1

–

12°C

HOLD

1

For additional details on optimizing ampliﬁcation, see Prior to Starting.
Appropriate temperature for P5/P7 Primer Mix (10X).
3
For the truncated adapter scheme detailed in Glenn et. al. 2019,use 55°C. Optimization may be required for other adapter/primer
conﬁgurations, see Prior to Starting.
1

2

Table 3. Recommended PCR cycle numbers by RNA
amount input into library prep

9.12 Incubate tubes at room temperature for at least
2 min before placing back on the magnet.

RNA input into Library
Preparation (ng)9.13 Leave tubes on the magnet for at least 2 min, or
until all beads have collected on the tube walls andthe solution is clear.

251 – 1000

8–9

101 – 250

10 – 1251 – 100

12 – 13

9.14 Carefully transfer 20 µL of each library-containing
supernatant to a new, labeled tube.

11 – 50

13 – 15

2.5 – 1016 – 18

Safe stopping point. Samples can be stored at 4°C
for <24 hours or frozen at -20°C for up to 4 weeks.

See
Table 3

PCR cycles to generate
10 – 50 nM library1

INSERT SIZE NOTE: An extra 2 PCR cycles are required when targeting
290 to 310 bp inserts using a 0.7X post-amplification cleanup(Step 11.2).

1

NOTE: An extra 2 PCR cycles are required whentargeting 290 to 310 bp inserts using a 0.7X postamplification cleanup (Step 11.2).

For Research Use Only. Not for use in diagnostic procedures.

10

Watchmaker mRNA Library Prep Kit

10.3 Assemble each ampliﬁcation reaction in the orderspeciﬁed below:
Component

Volume (µL)

Adapter-ligated library20

P5/P7 Primer Mix (10X)orUser-supplied primers1

5

Equinox Ampliﬁcation Master Mix (2X)25

1

See Prior to Starting for more information on user-supplied primers.

10.4 Mix on an appropriate vortex mixer (2,000 to
3,000 rpm) for 4 sec. Brieﬂy centrifuge to collect
all liquid in the bottom of the tubes.
10.5 Place tubes in the thermocycler (programmed in
Step 10.2) and initiate the program.
10.6 Once the program has completed, proceed
immediately to Post-ampliﬁcation Cleanup.
11. Post-ampliﬁcation Cleanup
11.1 Freshly prepare at least 0.4 mL of an 80% ethanol
solution for each reaction.
11.2 Vortex room temperature SPRI beads to thoroughly
mix. Add 50 µL (1X) of room temperature
resuspended beads to each ligation reaction.
INSERT SIZE NOTE: When targeting 290 – 310 bp
inserts, add 35 µL (a 0.7X ratio) of room
temperature resuspended beads to each ligation
reaction.
11.3 Mix on an appropriate vortex mixer (2,000 to
3,000 rpm) for 4 sec. Brieﬂy centrifuge to collect
all liquid in the bottom of the tubes.
11.4 Incubate the library-bead mixtures at room
temperature for at least 5 min to maximize libraryrecovery.
11.5 Place tubes on a magnet for at least 5 min, or until
all beads have been collected on the tube walls
and the solution is clear.

User Guide

11.7 Add 200 µL of freshly prepared 80% ethanol toeach tube, taking care to not disturb the beadpellet on the tube wall.
11.8 Incubate tubes at room temperature for at least
30 sec without removing them from the magnet
or disturbing the bead pellet. Carefully remove anddiscard the ethanol.
11.9 Repeat Steps 11.7 – 11.8 for a total of two washes.
OPTIONAL: Tubes can be brieﬂy centrifuged topull down excess ethanol prior to removing with a
P10 pipette.
11.10 Allow remaining ethanol to evaporate by allowing
the pellets to air dry for 3 – 5 min.
NOTE: Beads have been suﬃciently dried whencracks appear in the surface of the bead pellet
and no liquid is visible in the tubes. Residual trace
ethanol in the library ampliﬁcation reaction may
decrease performance.
11.11 Remove tubes from the magnet and carefully
resuspend each bead pellet in 22 µL of 10 mM
Tris-HCl, pH 8.0.
11.12 Incubate tubes at room temperature for at least
2 min before placing back on the magnet.
11.13 Leave tubes on the magnet for at least 2 min, or
until all beads have collected on the tube wall andthe solution is clear.
11.14 Carefully transfer 20 µL of each library-containing
supernatant to a new, labeled tube.
11.15 At this point, libraries are ready for quantiﬁcation,
normalization, pooling, hybrid capture, and/or
sequencing.
NOTE: We recommend quantifying libraries using
qPCR and analyzing quality and sizing using
capillary electrophoresis prior to preparing thelibraries for sequencing.

11.6 Carefully remove and discard the supernatantfrom each tube.

For Research Use Only. Not for use in diagnostic procedures.

11

Watchmaker mRNA Library Prep Kit

Appendix A: Amplification with Paramagnetic
Purification Beads
For some library preparation workflows, it may
be preferred or advantageous to perform PCR in
the presence of paramagnetic purification beads.
For example, in hybrid capture workflows, post-capture
amplification may be performed by adding PCR mix
directly to the capture beads with bound library,eliminating an elution step. Alternatively, libraries
may be eluted directly from SPRI beads in the PCR
reaction mixture.
We have found paramagnetic purification beads tofall into three categories of compatibility with PCR

User Guide

and sequencing based on their surface chemistry:
Group I—fullycompatible,
Group II—potentiallyinhibitory, and Group III—incompatible. While Group II
beads are inhibitory to non-optimized PCR systems,
Equinox Amplification Master Mix (2X) has been
optimized to allow amplification in the presence of bothGroup I and Group II beads, with no observable loss inperformance (e.g., efficiency, uniformity, or fidelity).
Equinox Amplification Master Mix (2X) and other library
amplification systems are not compatible with theextreme inhibition characterized by Group III beads.
Table A details the various paramagnetic bead
types evaluated.

Table A. Paramagnetic purification bead types
Bead type

VendorCatalognumberCompatibility/
amount tested*Group I (Tosyl-activated beads)
Dynabeads™ M280 Streptavidin

Thermo Fisher

11205D500 µgDynabeads MyOne™ Streptavidin T1

Thermo Fisher

65601

500 µg

Thermo Fisher

65305

500 µgVarious, incl. Beckman Coulter

A63882100 µL

Thermo Fisher

65001

500 µg

14305D500 µgGroup II (Carboxylic acid-activated beads)
Dynabeads M270 Streptavidin**
SPRI
Dynabeads MyOne Streptavidin C1

Group III (Not compatible with PCR)
Dynabeads M270 Carboxylic Acid

Thermo Fisher*Volume of slurry or mass of beads per 50 µL ampliﬁcation reaction.
**Used in xGen Hybridization and Wash Kit (Integrated DNA Technologies).

Revision History
VersionDescription

Date

1.0

• First protocol release

08/2023

1.1• Typographical error corrected in Step 2.7 to Capture Buffer instead of Final Wash Buffer.

08/2023

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www.watchmakergenomics.com
For Research Use Only. Not for use in diagnostic procedures.
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